Until about a decade ago, resolution in far-field light microscopy was thought to be limited to 200-250 nanometers in the focal plane, concealing details of sub-cellular structures and constraining its biological applications. Breaking this diffraction barrier by the seminal concept of stimulated emission depletion (“STED”) microscopy has made it possible to image biological systems at the nanoscale with light. STED microscopy and other members of reversible saturable optical fluorescence transitions (“RESOLFT”) family achieve a resolution greater than 10-fold beyond the diffraction barrier by engineering the microscope's point-spread function (“PSF”) through optically saturable transitions of the (fluorescent) probe molecules.
However, slow progress in 3D super-resolution imaging has limited the application of previously available techniques to two-dimensional (“2D”) imaging. The best 3D resolution until recently had been 100 nanometers axially at conventional lateral resolution. 4Pi microscopy achieved this through combination of two objective lenses of high numerical aperture, in an interferometric system. 4Pi microscopy was only recently shown to be suitable for biological imaging. Only lately the first 3D STED microscopy images have been published exceeding this resolution moderately with 139 nanometer lateral and 170 nanometer axial resolutions. While this represents a 10-fold smaller resolvable volume than provided by conventional microscopy, it is still at least 10-fold larger than a large number of sub-cellular components, for example synaptic vesicles.
Current understanding of fundamental biological processes on the nanoscale (e.g., neural network formation, chromatin organization) is limited because these processes cannot be visualized at the necessary sub-millisecond time resolution. Current biological research at the sub-cellular level is constrained by the limits of spatial and temporal resolution in fluorescence microscopy. The diameter of most organelles is below the diffraction limit of light, limiting spatial resolution and concealing sub-structure. Although recent developments have improved spatial resolution and even overcome the traditional diffraction barriers, comparable improvements in temporal resolution are still needed.
Particle-tracking techniques can localize small objects (typically less than the diffraction limit) in live cells with sub-diffraction accuracy and track their movement over time. But conventional particle-tracking fluorescence microscopy cannot temporally resolve interactions of organelles, molecular machines, or even single proteins, which typically happen within milliseconds. The spatial localization accuracy of single particles in a fluorescence microscope is approximately proportional to spatial resolution divided by the total number of detected fluorescence photons from the particle in the absence of background and effects due to finite pixel size. For longer acquisition times more signal can be accumulated, hence increased temporal resolution requires a trade-off of decreased spatial localization accuracy. For bright organelles containing a few hundred fluorescent molecules, (or future fluorescent molecules with increased brightness), sufficient signal can be accumulated quickly. However, especially for 3D localization where data acquisition is far more complicated than in 2D, technical constraints arising from axial scanning and/or camera readout times limit the recording speed, and therefore, the temporal resolution. Furthermore, results from axial scanning devices can taint detection processes and give at least a somewhat unclear or imprecise result.